Promoter activation by repositioning of RNA polymerase

Spo0A, a classical two-component-type response regulator in Bacillus subtilis, binds to a specific DNA sequence found in many promoters to repress or activate the transcription of over 100 genes. On the spoIIG promoter, one of the Spo0A binding sites, centered at position -40, overlaps a consensus -35 element that may also interact with region 4 of the sigma A (sigma(A)) Subunit of RNA polymerase. Molecular modeling corroborated by genetic evidence led us to propose that the binding of Spo0A to this site repositions sigma(A) region 4 on the promoter. Therefore, we used a chemical nuclease, p-bromoacetamidobenzyl-EDTA-Fe, that was covalently tethered to a single cysteine in region 4 of sigma(A) to map the position of sigma(A) on the promoter. The results indicated that in the absence of Spo0A, or A region 4 of the RNA polymerase was located near the -35 element sequence centered at position -40. However, in the presence of Spo0A, or A region 4 was displaced downstream from the -35 element by 4 bp. These and other results support the model in which the binding of Spo0A to the spoIIG promoter stimulates promoter utilization by repositioning prebound RNA polymerase and stabilizing the repositioned RNA polymerase-promoter complex at a new position that aligns sigma(A) region 2 with the -10 region sequences of the promoter, thus facilitating open complex formation.